We propose to determine how DNA methylation patterns are initiated and maintained in cells of higher organisms. To do this we have isolated two types of methylases, HpaII methylase from Hemophilus parainfluenza which methylates the inner cytosines in the CCGG sites and a "CG" methylase from mammalian cells. We have cloned into the E. coli plasmid BR322 the EcoRI methylase gene which methylates the inner adenines in the GAATTC sites. By methylating the DNA of SV40 and infecting cells with this DNA we hope to learn whether a pattern of methylation can be maintained in a lytic infection or in the integrated state of a viral DNA. We will also inject plasmids into the unfertilized eggs of Xenopus laevis as assay for ability to replicate and to initiate or maintain a pattern of methylation. We have determined that only plasmids with a DNA replication origin from frog or higher vertebrate cells will replicate in the unfertilized frog egg. Two complete rounds of replication have been shown to occur. The maintenance of the adenine methylation does not occur since the frog DNA does not normally contain N6-methyl adenine, but we hope to learn if 5-methyl cytosine methylation can be maintained in CG doublets -- some of which are normally methylated in frog DNA.